The responses of vertebrate neurones to glutamate involve at least three receptor types. One of these, the NMDA receptor (so called because of its specific activation by N-methyl-D-aspartate), induces responses presenting a peculiar voltage sensitivity. Above resting potential, the current induced by a given dose of glutamate (or NMDA) increases when the cell is depolarized. This is contrary to what is observed at classical excitatory synapses, and recalls the properties of 'regenerative' systems like the Na+ conductance of the action potential. Indeed, recent studies of L-glutamate, L-aspartate and NMDA-induced currents have indicated that the current-voltage (I-V) relationship can show a region of 'negative conductance' and that the application of these agonists can lead to a regenerative depolarization. Furthermore, the NMDA response is greatly potentiated by reducing the extracellular Mg2+ concentration [( Mg2+]o) below the physiological level (approximately 1 mM). By analysing the responses of mouse central neurones to glutamate using the patch-clamp technique, we have now found a link between voltage sensitivity and Mg2+ sensitivity. In Mg2+-free solutions, L-glutamate, L-aspartate and NMDA open cation channels, the properties of which are voltage independent. In the presence of Mg2+, the single-channel currents measured at resting potential are chopped in bursts and the probability of opening of the channels is reduced. Both effects increase steeply with hyperpolarization, thereby accounting for the negative slope of the I-V relationship of the glutamate response. Thus, the voltage dependence of the NMDA receptor-linked conductance appears to be a consequence of the voltage dependence of the Mg2+ block and its interpretation does not require the implication of an intramembrane voltage-dependent 'gate'.

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The established view in cellular biology dictates that the cellular internalization of hydrophilic macromolecules can only be achieved through the classical endocytosis pathway. However, in the past five years several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides have been used successfully for the intracellular delivery of macromolecules with molecular weights several times greater than their own. Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques because it is efficient for a range of cell types, can be applied to cells en masse and has a potential therapeutic application.

Neural circuits are shaped by experience in early postnatal life. Distinct GABAergic connections within visual cortex determine the timing of the critical period for rewiring ocular dominance to establish visual acuity. We find that maturation of the parvalbumin (PV)-cell network that controls plasticity onset is regulated by a selective re-expression of the embryonic Otx2 homeoprotein. Visual experience promoted the accumulation of non-cell-autonomous Otx2 in PV-cells, and cortical infusion of exogenous Otx2 accelerated both PV-cell development and critical period timing. Conversely, conditional removal of Otx2 from non-PV cells or from the visual pathway abolished plasticity. Thus, the experience-dependent transfer of a homeoprotein may establish the physiological milieu for postnatal plasticity of a neural circuit.

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Specific transfer of Otx2 homeoprotein into GABAergic interneurons expressing parvalbumin (PV) is necessary and sufficient to open, then close, a critical period (CP) of plasticity in the developing mouse visual cortex. The accumulation of endogenous Otx2 in PV-cells suggests the presence of specific Otx2 binding sites. Here, we find that perineuronal nets (PNNs) on the surface of PV-cells permit the specific, constitutive capture of Otx2. We identify a 15 amino-acid domain containing an arginine-lysine doublet (RK-peptide) within Otx2, bearing prototypic traits of a glycosaminoglycan (GAG) binding sequence that mediates Otx2 binding to PNNs and specifically Chondroitin sulfate D and E with high affinity. Accordingly, PNN hydrolysis by Chondroitinase ABC reduces the amount of endogenous Otx2 in PV-cells. Direct infusion of RK-peptide similarly disrupts endogenous Otx2 localization to PV-cells, reduces PV and PNN expression and reopens plasticity in adult mice. The closure of one eye during this transient window reduces cortical acuity and is specific to the RK motif, as an AA variant or scrambled peptide fail to reactivate plasticity. Conversely, this transient reopening of plasticity in the adult restores binocular vision in amblyopic mice. Thus, one function of PNNs is to facilitate the persistent internalization of Otx2 by PV-cells to maintain CP closure. The pharmacological use of the Otx2 GAG-binding domain offers a novel, potent therapeutic tool with which to restore cortical plasticity in the mature brain.

During the past fifteen years, a variety of peptides have been characterized for their ability to translocate into live cells. Most are efficient vectors that can internalize hydrophilic cargoes, and so provide a valuable biological (and potentially therapeutic) tool for targeting proteins into cells. Furthermore, translocation of cell-permeable peptides across the plasma membrane and their subsequent access to the cytosol, even when fused to large hydrophilic proteins, is challenging the perception of the plasma membrane as an impermeable barrier.

We synthesized the 60-amino acid polypeptide corresponding to the sequence of the Drosophia antennapedia gene homeobox. This peptide (pAntp) recognized the consensus motif for binding to the promoter region ofHox-1.3. pAntp mechanically introduced into mammalian nerve cells provoked a dramatic morphological differentiation of the neuronal cultures. Moreover, pAntp directly added to already differentiated neuronal cultures penetrated the cells and further augmented their morphological differentiation. Examination of live and fixed neurons in classical and confocal fluorescence microscopy demonstrated that pAntp was captured at all regions of the nerve cells and accumulated in the nuclei. In addition, the effect of pAntp on neurite extension was blocked in the presence of the protein synthesis inhibitor cycloheximide. Thus, our results demonstrate that neurons possess an efficient uptake system for the antennapedia homeobox peptide and suggest that binding of pAntp to consensus motifs present in nerve cell nuclei influences neuronal morphogenetic programs.Studies on developmental mutants in Drosophila have demonstrated that several DNA binding proteins encoded by homeotic genes are endowed with morphogenetic functions (1, 2). In Drosophila at least, the expression of specific homeotic genes is responsible for the formation of cell assemblies exhibiting precise and defined morphologies. The DNA binding properties ofthese proteins is due to a sequence of about 60 amino acids called the homeobox.The homeobox sequences have been highly conserved during evolution and genes containing homeobox sequences are present in all vertebrates, including mammals (3, 4). In vertebrates, as in Drosophila, homeobox gene expression is not limited to the period during which the general features of body organization are established. In particular, a number of these genes are expressed in the nervous system rather late during development and, in some cases, through adulthood (5-7).In vitro studies have shown that the regulatory effect of homeotic proteins on gene expression depends on the specific binding of the homeobox domain to consensus DNA sequences found in the promotors or enhancers of several reporter genes and homeobox-containing genes (8-10). It has also been clearly demonstrated that the 60-amino acid homeobox polypeptide alone, isolated from the flanking regions, which are necessary for the activation or repression of transcription, has per se a high affinity for such consensus motifs (11).Thus, one possible way to study directly, within the live cell, the role of a homeotic protein family, as defined by the primary structure of the homeobox (e.g., antennapedia-or engrailed-like) and by the recognition of identical binding sites, on neural development would be to synthesize and inject the 60-amino acid homeotic polypeptides into the cells. In fact, such peptides could act as competitive inhibitors of endogeneous homeotic proteins with similar binding specificities.In the experiments presented herein we have used this...